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1 Original Article Diverse Genetic Subtypes of Hiv-1 Among Female Sex Workers in Ibadan, Nigeria

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1 Original Article Diverse Genetic Subtypes of Hiv-1 Among Female Sex Workers in Ibadan, Nigeria ORIGINAL ARTICLE AFRICAN JOURNAL OF CLINICAL AND EXPERIMENTAL MICROBIOLOGY. JANUARY 2014 ISBN 1595-689X VOL15 No.1 AJCEM/1322 http://www.ajol.info/journals/ajcem COPYRIGHT 2014 http://dx.doi.org/10.4314/ajcem.v15i1.1 AFR. J. CLN. EXPER. MICROBIOL 15(1): 1-8 DIVERSE GENETIC SUBTYPES OF HIV-1 AMONG FEMALE SEX WORKERS IN IBADAN, NIGERIA *1Fayemiwo, S. A., 2Odaibo, G. N. 3 Sankale , J.L., 1Oni, A.A., 1Bakare, R. A., 2Olaleye, O. D. and 4Kanki, P. Departments of 1Medical Microbiology and 2Virology, College of Medicine, University College Hospital, Ibadan, Nigeria and 3A.P.I.N., Harvard School of Public Health, Boston, MA , USA. Running title: Genetic subtypes of HIV-1 among female sex workers in Nigeria. Keywords: Diverse, HIV, subtypes, Female Sex workers and Vaccine *Correspondence: Fayemiwo, S. A., Department of Medical Microbiology and Parasitology, College of Medicine, University of Ibadan, Ibadan. Nigeria. E-mail address: [email protected] ABSTRACT Background: Genetic diversity is the hallmark of HIV-1 infection. It differs among geographical regions throughout the world. This study was undertaken to identify the predominant HIV-1 subtypes among infected female sex workers (FSWs) in Nigeria. Methods: Two hundred and fifty FSWs from brothels in Ibadan Nigeria were screened for HIV antibody using ELISA. All reactive samples were further tested by the Western Blot Techniques. Peripheral Blood Mononuclear Cells (PBMCs) were separated from the blood samples of each subject. Fragments of HIV Proviral DNA was amplified and genetic subtypes of HIV-1 was determined by direct sequencing of the env and gag genes of the viral genome followed by phylogenetic analysis . Results: The age of the FSWs ranged from 15 to 55 years old (Mean = 25.8years, SD =3.74). Majority were Nigerians while others (1.6 %) were from neighboring West Africa countries. Four ( 1.6% ) of the FSWs were active for less than one year as sex workers, and the mean length of sex work was 2.80 years ( Range = 1.0 – 15.0 years ). Sixty-four (25.6%) of the 250 CSWs were positive for HIV-1 while 7 (2-8%) had dual infections to HIV-1 / HIV-2. Among the 34 HIV-1 strains characterized by sequencing, 19 (55-9%) were subtype G, 9 (26.5%) CRF02_A/G, 3 (8.8%) CRF06_cpx while 1 (2.9%) each were identified as subtype C, CRF01_A/E and CRF09_cpx respectively. Nineteen (55.9%) of the FSWs with subtype G had been active in the sex work for between one to five years. The youngest of the HIV -1 infected FSWs with sexual activity of less than a year had subtype G strain. There is a significant probability that infection with this subtype occurred with a short incubation period (p< 0.05). Conclusion: This study showed a wide range of HIV- 1 subtypes among FSWs in Nigeria. The situation poses serious challenge for the design of HIV vaccine candidate for use in Nigeria. INTRODUCTION Female sex workers (FSWs) have been at high-risk of infection since the beginning of the HIV epidemic all The Acquired Immune Deficiency Syndrome (AIDS) over the world. Multiple sex partners, irregular epidemic has exacted a terrible toll in terms of loss of condom use, and co-infection with other sexual life and decreased quantity of life worldwide, transmitted infections (STIs) are the principal risk especially in Africa, where 70% of deaths from HIV-1 factors for HIV infection among FSWs (7). Between infection have occurred (1). 1995 and 2003 the prevalence rose from 6.4% in STD patients (8) to 34.3% in FSWs (9). The penetration into The first case of AIDS in Nigeria was reported in the general population cannot be over emphasized. 1986. From 2 cases in 1986, it has been estimated that 4,050,000 HIV infections have occurred in Nigeria 2, In women, the glandular epithelia have HIV in the and it has spread extensively to the rural areas of the zone of transformation between the columnar and Country (3, 4, 5). squamous cell of the cervix (10). While in men, HIV is detectable in seminal cells, some sperm cells and their In Africa, infection has been mainly by heterosexual precursors (11). Because of high incidence of sexually intercourse, with Female sex workers (FSWs), long transmitted infections (STIs) in African sex workers, distant truck drivers and migrant labour serving as immunologic stimulations to this infection may be vehicles of spread (5). higher than among the general African population (12). 1 HIV-1 and HIV-2 show heterogeneity among isolates MATERIALS AND METHODS (13). On the basis of the phylogenetic analysis of the env and gag genes, HIV-1 has been divided into three Study population and Sample collection distinct groups (M, O, & N.), all of which cause AIDS in infected individuals. Group M (“For Major”) A cross- sectional seroprevalence study was comprises the greatest majority of HIV-1 infections conducted in a population at high risk for HIV worldwide and has been further subdivided into infection from November 2002 and July 2003 in subtypes and sub subtypes termed A-D, F.H, J, K, Ibadan, South Western Nigeria. Only those subjects and Circulating Recombinants Forms; CRF 01 to 34 who were willing to participate were provided (13,14). Whereas several HIV-1 subtypes co-circulate written informed consent, enrolled, and sampled. A in many parts of the world, some regions seem to total of two hundred and fifty Female sex workers have predominant subtypes. Subtype B is the most from various brothels in Ibadan that had been in the predominant form in North America and Europe profession for at least one month and duly signed while subtype E is highly prevalent in part of South informed consent were recruited into the study. The East Asia (where B is the minority form) (15). study protocol was approved by the University of Subtypes A, C, and D are the most common subtypes Ibadan/ University College Hospital (UCH) ethical in Africa. In Mali 80% of the HIV-1 infected sex review committee. Semi-structured questionnaire was workers were subtype A, 10% belong to subtype G administered to each of the FSW who gave informed and the minority belong to subtypes C and D (16), consent, as an instrument to elicit base line while in Kenya 56% of the infected subjects had information on their demographic characteristics and subtypes A while others have subtypes C and D with reproductive health history. other recombinants circulating forms (17). Ten millimeters of EDTA – anti coagulated blood In Nigeria, the first HIV-1 strain that was partially samples were collected from the recruited female sex characterized was found to be subtype A (18). In workers. These samples were transported on an ice recent time , we have co-circulation of multiple parks in an insulated box to the Diagnostic subtypes including, A, B, C, D, E and other groups in Laboratory, Virology Department, University College Nigeria with subtype C being the most prevalent (19). Hospital, Ibadan for processing. Plasma or sera were subsequently separated and stored in aliquots and The city of Ibadan, which is acclaimed to be the maintained at -70 oc until tested. Clinical examinations largest city in West Africa, and capital of Oyo State, is of the lower genito-urinary tracts were carried out for a major commercial centre with long distant transport signs of infections such as vaginal discharge, workers, migrant labour workers, civil servants and endocervical discharge, genital ulcer diseases and students of higher institutions which have sex with genital mass (genital warts and bartholin’s cyst). multiple female sex workers with risk of acquiring HIV infection. The occurrences of these multiple variants of HIV in Antibody Screening and Confirmation different geographical regions raise the possibility All the sera or plasma separated from the samples that future vaccine strategies may have to consider collected were screened using Enzyme Linked polyvalent constructs. The effective evaluation of Immunosorbent Assay (ELISA) techniques with vaccine candidates will depend on thorough commercial available kits (GENSCREEN PLUS HIV surveillance and characterization of viral variants by Bio-Rad in France). All reactive samples were (20). confirmed by Western blot assay (New LAV BLOT 1 Since HIV infection most often results from by Bio-Rad, in France). heterosexual intercourse, female sex workers in DNA Extraction and Polymerase Chain Reactions various brothels in Ibadan are likely to be the source of detection of new subtypes that might have been Qiagen kit was used for the extraction of DNA from introduced into the country and likely point for rapid the Peripheral Blood Mononuclear Cells separated spread of these different subtypes. Therefore this from the whole blood of the FSWs that had been study was aimed at providing information on the previously stored and maintained at -70 0C. The sero-prevalence of HIV among female sex workers in Qiagen genomic DNA purification procedure is Ibadan and also to identify the predominant subtypes designed to isolate chromosomal DNA 20-150kb of HIV-1 among them. directly from the whole blood, buffy coat and cultured cells. Genomic DNA is eluted in a high salt buffer, concentrated and desalted by isopropanol 2 precipitation. About 5ul of the genomic DNA of the RESULTS confirmed ELISA reactive blood samples were combined with primers: WTI (KK30) and WT2 A total of 250 Female sex workers (FSWs) from (KK40), buffers, nucleotides , taq polymerase and various brothels in Ibadan were enrolled and distilled water in a micro centrifuge tubes. Positive screened for HIV antibody seropositivity and and negative controls were included in each run. The predominant HIV-1/2 subtypes was determined total volume in each tube was made up to 50 ul in the during the period of study.
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